1. Field of the Invention
The present invention relates to a method for microscopically detecting a nucleic acid molecule hybridized with a probe.
2. Description of the Related Art
In detecting a nucleic acid having a specific sequence, use is made of a method using a probe labeled with a radioisotope or fluorescent substance, a method of measuring the activity of an enzyme immobilized onto a probe, and a method for enzymatically amplifying a specific sequence, such as in PCR (polymerase chain reaction) (see EPO192168A2, WO91/14788, U.S. Pat. No. 4,734,363 etc.). These methods include the step of amplifying signals by certain means and raise the problem that this amplification step or a preliminary step therefor is so cumbersome that a longer time is required.
In addition, in cases where a probe labeled with a radioisotope, fluorescent substance or enzyme is used, while the amplification of the desired signals is accomplished through the labeled prove which has been bound to the target sequence, noises are also amplified through the probe which has been non-specifically bonded to a solid phase carrier having a sample and the probe immobilized thereon, thereby bringing about the problem of adverse influence on detection accuracy. To prevent this problem, it is necessary to take measures such as vigorous washing after hybridization, inclusion of other DNA at the time of hybridization, etc. However, even if such measures are taken, it is not possible to prevent nonspecific binding completely, and if the concentration of the target nucleic acid molecule is low, higher noises are caused by nonspecifically bound nucleic acid molecules, which may result in failing to detect the signals from specifically bound nucleic acids. Further, because these additional procedures make the inherently complicated experimental operation further cumbersome, there is the drawback of increases in the labor and time required for the experiment. In addition, probe-labeling operation itself also requires cumbersome procedures. Further, if a radioisotope is used, there are potential problems that a place for conducting the experiment is limited, and qualification as the experimenter is required, etc.
On the other hand, if the PCR method is used for detecting DNA, it is disadvantageous that the step of enzymatically amplifying DNA needs a longer time, and further steps of fractionating DNA by gel electrophoresis etc. is required after the amplification step. Further, a spectrophotometer, fluorescence spectrophotometer or fluorescence microscope is used for detection of signals of labeled nucleic acid molecules. In these detection systems, the target nucleic acid molecule hybridized with the probe is detected by detecting the activity of the enzyme or the fluorescence of the fluorescent substance with which the probe has been labeled. Accordingly, these detection systems suffer from the problem that the specific binding between the nucleic acid probe and the target nucleic acid molecule cannot be distinguished from the nonspecific binding of the nucleic acid probe to other nucleic acids than the target nucleic acid molecule or to the solid carrier surface.
As described above, the conventional methods of detecting nucleic acid make use of a labeled nucleic acid probe, so the step of removing the unbound nucleic acid probe by washing is required to eliminate background noises from data at the time of measurement. Further, the subject to be detected by labeling is the presence of the labeled substance itself, but not the binding itself of the probe to the target nucleic acid molecule. Therefore, there is no means of distinguishing noises caused by nonspecific binding from the signal of specific binding. As a result, if the concentration of the target nucleic acid molecule is low, noises caused by nonspecific binding are higher than the signal from specific binding, which may lead to the erroneous conclusion that the target nucleic acid molecule is not present in the sample. On the other hand, if the nonspecifically bound nucleic acid probe remains due to insufficient washing, the conclusion that the target molecule is present in the sample may be derived although the target nucleic acid is not actually present. Furthermore, because this nonspecific binding cannot be eliminated completely, it cannot be determined whether the signals detected are due to an increase in nonspecific binding resulting from insufficient washing etc. or due to specific binding when the signals are slightly higher than noises caused by nonspecific binding. Thus, such signals are considered as pseudo-positive data and it cannot be determined, in some cases, whether these data are inherently positive or negative.